Stimulation of cells with a variety of cytokines as well as transformation with oncogenic versions of growth factors (GF), GF receptors, and tyrosine kinase oncogenes results in phosphorylation of Raf-1 protein, resulting in a mobility shift and increased kinase activity. We have demonstrated recently that the kinase activity of another Raf family member, B-raf, is regulated in a ligand-dependent fashion in PC12 pheochromocytoma cells when induced to differentiate by nerve growth factor. Ras family GTP binding proteins have been implicated in signaling pathways that result in proliferation and differentiation. Ras appears to function upstream of Raf-1, as judged from work with kinase activating mutants of Raf-1 that overcome a block to cellular Ras function and inhibitory mutants of Raf-1, as well as Raf-1 antisense constructs, which block growth induction and transformation by oncogenic versions of Ras. These combined data suggest that Ras may control phosphorylation-activation of Raf by GF receptors. We have used a dominant negative mutant of Ras, M17 Ras, to analyze whether Ras is required for induced phosphorylation and activation of Raf- 1 kinase in NIH/3T3 cells and Raf-1 as well as B-Raf-kinase in PC12 pheochromocytoma cells. Our results show that (1) M17Ras blocks kinase activation of Raf-1 in NIH/3T3 cells after stimulation with serum, TPA, PDFG, as well as EGF. (2) Similarly, activation of Raf-1 and B-Raf after NGF, EGF, PDGF, and TPA stimulation is blocked in PC12 cells expressing M17 Ras. (3) Tryptic phosphopeptide mapping of Raf-1 and B-Raf demonstrates that all GF-sensitive spots were suppressed by mutant Ras expression, suggesting that Ras controls the activity of Raf kinase kinases or that it regulates Raf-1 and B-Raf autophosphorylation. (4) GF receptor autophosphorylation and PLC-gamma1 phosphorylation in the same cells were unaffected by M17 Ras, demonstrating that a block of Ras function does not generally interfere with the signaling from GF receptors. Furthermore, a synergistic interaction between Ras and Raf was demonstrated in experiments using cotransfection of both proto-oncogenes into NIH3T3 cells and assay for transformation and transcription activation.